Antibiotic GE 2270 factors A1, A2, A3 and H

ABSTRACT

The present invention is directed to new antibiotic substances denominated antibiotic GE 2270 factors A 1 , A 2 , A 3  and H, the addition salts thereof, the pharmaceutical compositions thereof and their use as medicaments, particularly in the treatment of infectious diseases involving microorganisms susceptible to them.

This is a continuation of application Ser. No. 08/135,862, filed Oct.13, 1993, which is a continuation of application Ser. No. 08/010,504,filed Jan. 28, 1993, now abandoned, which is a continuation ofapplication Ser. No. 07/895,507, filed Jun. 8, 1992, now abandoned,which is a divisional of application Ser. No. 07/547,647, filed Jul. 2,1990, which is U.S. Pat. No. 5,139,778, which issued on Aug. 18, 1992,herein incorporated by reference.

The present invention is directed to new antibiotic substancesdenominated antibiotic GE 2270 factors A₁, A₂, A₃ and H, the additionsalts thereof, the pharmaceutical compositions thereof and their use asmedicaments, particularly in the treatment of infectious diseasesinvolving microorganisms susceptible to them.

The compounds of the invention are also active as growth promotantagents in animals, such as poultry, swine, ruminants, etc.

Another object of this invention is a process for preparing antibioticGE 2270 factors A₁, A₂, A₃ or H by selective transformation ofantibiotic GE 2270 factor A. This substance is, in turn, prepared byculturing a sample of Planobispora rosea ATCC 53773 or a producingvariant or mutant thereof and isolating the desired antibiotic substancefrom the mycelium and/or the fermentation broth. Planobispora rosea ATCC53773 was isolated from a soil sample and deposited on Jun. 14, 1988with the American Type Culture Collection (ATCC), 12301 Parklawn Drive,Rockville, Md. 20852 Maryland, U.S.A., under the provisions of theBudapest Treaty.

The strain has been accorded accession number ATCC 53773.

Antibiotic GE 2270 factor A and Planobispora rosea ATCC 53773 aredescribed in European Patent application Publication No. 359062.

In view of the similarities between the antimicrobial activity ofantibiotic GE 2270 factor A₁, A₂, A₃ and H and the correspondingpharmaceutically acceptable salts, when available, in the presentapplication when dealing with the biological properties of antibiotic GE2270 factors A₁, A₂, A₃ or H, also the corresponding salts are includedand vice versa, when dealing with the biological properties of apharmaceutically acceptable addition salt of antibiotic GE 2270 factorA₁, A₂, A₃ or H, also the corresponding "non-addition salt" form isencompassed.

In particular, antibiotic GE 2270 factor A₃ can form base addition saltsaccording to the usual procedures.

Representative examples of these bases are: alkali metal ofalkaline-earth metal hydroxides such as sodium, potassium, calcium,magnesium, and barium hydroxide; ammonia and aliphatic, alicyclic oraromatic organic amines such as methylamine, dimethylamine,trimethylamine, and picoline. Also the salts with basic aminoacids orderivatives thereof such as arginine, lysine or ornithine areencompassed by the present definition of base addition salts.

The transformation of the "non-salt" compounds of the invention into thecorresponding addition salts, and the reverse, i.e. the transformationof an addition salt of a compound of the invention into the non-saltform, are within the ordinary technical skill and are encompassed by thepresent invention.

A base addition salt of antibiotic GE 2270 factor A₃ can be prepared forinstance by dissolving or suspending the non-salt form in an aqueoussolvent and adding a slight molar excess of the selected base. Theresulting solution or suspension is then lyophilized to recover thedesired salt.

In case the final salt is unsoluble in a solvent where the non-salt formis soluble, it is recovered by filtration from the organic solution ofthe non-salt form after addition of the stoichiometric amount of aslight molar excess of the selected base.

The non-salt form can be prepared from a corresponding base saltdissolved in an aqueous solvent which is then neutralized to free thenon-salt form.

When following the neutralization the elimination of the excess of acidor base is necessary, a common desalting procedure may be employed.

For example, column chromatography on silanized silica gel,non-functionalized polystyrene, acrylic and controlled pore polydextraneresins (such as Sephadex® LB 20) or activated carbon may be convenientlyused. After eluting the undesired salts with an aqueous solution, thedesired product is eluted by means of a linear gradient or astep-gradient of a mixture of water and a polar or apolar organicsolvent, such as acetonitrile/water from 50:50 to about 100%acetonitrile.

The compounds of the invention are prepared from antibiotic GE 2270factor A under selective chemical transformation conditions.

More particularly, antibiotic GE 2270 factors A₁, A₂ and A₃ are preparedby antibiotic GE 2270 factor A under selective hydrolysis conditions,while by selective cleavage under reducing conditions antibiotic GE 2270factor H is obtained from antibiotic GE 2270 factor A.

Generally, the above mentioned hydrolytic conditions involve the use ofmixtures of buffered or unbuffered aqueous acid media and polar organicsolvents. The reaction temperature varies depending on such factors asthe strength and the concentration of the acid employed, and isgenerally comprised between -10° C. and 90° C. Also the reaction timevaries considerably depending on the other parameters such astemperature and acid strength and concentration; generally it may varyfrom a few minutes to several hours.

In general, when milder hydrolysis conditions are employed, e.g. shortertime and lower temperature or lower acid strength or concentration,antibiotic GE 2270 factor A₁ is normally obtained, while under strongerconditions antibiotic GE 2270 factor A₂ is obtained. To obtainantibiotic GE 2270 factor A₃ under acid hydrolyric conditions still moredrastic conditions are necessary.

However, since the reaction course can be monitored according to theusual procedures, e.g. TLC or HPLC, by following the disappearance ofthe starting material and/or appearance of the final products, theskilled technician is capable of deciding when the reaction can beconsidered as complete and the recovery procedure may be started.

Examples of buffered or unbuffered aqueous acidic media are aqueoussolutions or suspensions of mineral or organic acids such as hydrogenhalides, e.g. hydrogen chloride, bromide or iodide; phosphoric acids;sulfuric acid; (C₁ -C₆)aliphatic, halogenated(C₂ -C₅)aliphatic or arylicacids; (C₂ -C₆)alkylsulfonic or arylsulfonic acids; cationic exchangeresins in the acid form, and the products of partial salification ofpolyprotonic acids, i.e., salts having acidic reaction in water, e.g. analkali-metal hydrogen phosphate or dihydrogen phosphate, alkali metalhydrogen sulfate and the like. Examples of alkali-metals are sodium andpotassium.

Representative examples of the (C₁ -C₆)aliphatic acids mentioned aboveare formic acid, acetic acid, propionic acid, butyric acid, isobutyricacid, valeric acid, isovaleric acid, trimethylacetic acid, and the like.

Representative examples of the halogenated(C₂ -C₅)aliphatic acidsmentioned above are mono- or poly- chloro, bromo or iodo aliphatic acidssuch as fluoroacetic acid, chloroacetic acid, difluoroacetic acid,dichloroacetic acid, trifluoroacetic acid, trichloroacetic acid,pentafluoropropionic acid, 2,2,3,4,4,4-hexafluorobutyric acid,heptafluorobutyric acid, and the like.

Representative examples of arylic acids are benzoic acid and mono- orpoly- substituted benzoic acids such as chlorobenzoic acid,methylbenzoic acid, phthalic acid, terphthalic acid, benzylacetic acidand the like.

Representative examples of the sulfonic acids mentioned above are:methanesulfonic acid, trifluoromethanesulfonic acid, p-toluenesulfonicacid, cyclohexanesulfonic acid, camphorsulfonic acid, alpha- andbeta-naphthalenesulfonic acid.

The "cationic exchange resins" mentioned above are commonly known andcommercially available resins such as sulfonated styrenic andstyrene-divinyl benzenic resins in the acid form.

Suitable organic solvents as mentioned above are such that:

a) they may at least partially solubilize the starting materials;

b) the products, once obtained, either separate or may be separatedaccording to usual techniques, and

c) in any case, they do not unfavorably interfere with the reactioncourse.

Examples of said organic polar solvents are cyclic oxygen containingaliphatic solvents such as dioxane (i.e.diethylenedioxide),tetrahydrofuran and the like; lower alkanols; phenyl substituted loweralkanols; lower alkyl carboxamides; lower alkyl sulfoxamides; loweralkyl phosphoramides; lower alkyl sulfoxides and lower alkyl sulfones,and the like, and mixtures thereof. The term "lower alkyl" as usedabove, represents preferably alkyl groups of 1 to 6 carbon atoms.Examples of lower alkanols are C₁ -C₆ alkanols such as methanol,ethanol, propanol, 1-methylethanol, butanol and 2-methylpropanol.

Examples of phenyl substituted lower alkanols are the following: benzylalcohol, m-chlorobenzyl alcohol, o-fluorobenzyl alcohol, m-fluorobenzylalcohol, p-fluorobenzyl alcohol, m-methylbenzyl alcohol, m-methoxybenzylalcohol, o-ethoxybenzyl alcohol, m-butoxybenzyl alcohol,p-tert.butoxybenzyl alcohol, p-tert.butylbenzyl alcohol, phenethylalcohol, o-chlorophenethyl alcohol, m-chlorophenetyl alcohol,o-methoxyphenethyl alcohol, m-methoxyphenethyl alcohol,o-propylphenethyl alcohol, o-ethoxyphenethyl alcohol, p-fluorophenethylalcohol, p-bromophenethyl alcohol, o-propoxyphenethyl alcohol,o-butoxyphenethyl alcohol, 1-(p-isopropylphenyl)ethanol,3-phenyl-1-propanol, 2-phenyl-1-propanol, 4-phenyl-1-butanol and3-phenyl-1-butanol.

Examples of lower alkyl carboxamides are dimethylformamide,diethylformamide and the like. A preferred lower alkyl sulfoxide isdimethyl sulfoxide, a preferred lower alkyl sulfone is dimethylsulfoneand a preferred lower alkyl phosphoramide is hexamethyl phosphoramide.

A preferred embodiment of the invention is represented by a process forpreparing antibiotic GE 2270 factor A₁ which comprises contactingantibiotic GE 2270 factor A with a buffered or unbuffered acid medium inthe presence of a polar organic solvent at a temperature between -10° C.and 50° C., and preferably between 4° C. and 25° C. The reaction time,which varies considerably depending on the other specific reactionparameters, is, in this case, generally between 5 min and 16 h.

Another preferred embodiment of the invention is represented by aprocess for preparing antibiotic GE 2270 factor A₂ which comprisescontacting antibiotic GE 2270 factor A or factor A₁ with a buffered orunbuffered acid medium in the presence of a polar organic solvent at atemperature between 40° C. and 90° C., and preferably between 50° C. and70° C. The reaction time, which varies considerably depending on theother specific reaction parameters, is, in this case, generally between12 h and 24 h.

In the case of the conversion of factor A₁ into factor A₂ under theabove conditions, it appears that ammonia is lost by the molecule of thestarting material.

A further preferred embodiment of the invention is represented by aprocess for preparing antibiotic GE 2270 factor A₃ which comprisescontacting antibiotic GE 2270 factor A with a buffered or unbufferedacid medium in the presence of a polar organic solvent at a temperaturebetween 40° C. and 90° C., and preferably between 65° C. and 80° C. Thereaction time, which varies considerably depending on the other specificreaction parameters, is, in this case, generally between 8 h and 48 h.

In this case however, the conversion yields are generally low (5-15%molar yield) even by using strong reaction conditions.

A preferred method for preparing antibiotic 2270 factor A₃ withacceptable yields and purity is represented by the treatment ofantibiotic GE 2270 factor A₂ under basic hydrolytic conditions in thepresence of polar organic solvents. Examples of polar organic solventsare as reported above, while the basic conditions can be imparted by anyaqueous basic solution or suspension.

Examples of base that can conveniently be employed are inorganic ororganic bases such as alkali-metal or earth-metal hydroxides; saltshaving basic reaction in water such as alkali-metal carbonates orbicarbonates; ammonia and aliphatic or aromatic amines such as alkylamines (e.g. dimethylamine, diethylamine, triethylamine, trimethylamine)and picolines. Examples of alkali-earth metals are calcium andmagnesium.

The following specific reaction conditions are given simply to furtherillustrate the process of the invention.

In the following tables:

"R.T." means "room temperature" i.e. approximately 15° C.-25° C.

"EtOH" means "ethanol"

"TFA" means "trifluoroacetic acid"

"AcOH" means "acetic acid"

"Dowex" 50Wx2 (H+) is a cation exchange resin in the acid form

"TEA" means "triethylamine".

Specifically preferred reaction conditions for the preparation ofantibiotic GE 2270 factor A₁ from factor A are the following:

    ______________________________________                                        Temperature                      Polar                                        °C.                                                                             Time      Acid medium   solvent                                      ______________________________________                                        R.T.      5 min    0.2M HCl      EtOH/H.sub.2 O                                                                9/1                                          R.T.     15 min    TFA/dioxane/H.sub.2 O                                                         1/4/5                                                      R.T.     15 min    0.5N H.sub.2 SO.sub.4                                                                       Dioxane/H.sub.2 O/                                                            1/1                                          R.T.     15 min    0.25M H.sub.3 PO.sub.4                                                                      Dioxane/H.sub.2 O/                                                            1/1                                          50       overnight 0.5M NH.sub.4 Cl                                                                            Dioxane/H.sub.2 O/                                                            1/1                                          ______________________________________                                    

Specifically preferred reaction conditions for the preparation ofantibiotic GE 2270 factor A₂ from factor A or A₁ or a mixture thereofare the following:

    ______________________________________                                        Temperature                      Polar                                        °C.                                                                             Time      Acid medium   solvent                                      ______________________________________                                        60° C.                                                                          24 h      AcOH/EtOH (95%)                                            80° C.                                                                          overnight AcOH/H.sub.2 O, 1/1                                        50° C.                                                                          overnight TFA/dioxane/H.sub.2 O,                                                        1/4/5                                                      50° C.                                                                          overnight 0.25M H.sub.3 PO.sub.4                                                                      Dioxane/H.sub.2 O/                                                            1/1                                          50° C.                                                                          overnight 0.5 M NH.sub.4 Cl                                                                           Dioxane/H.sub.2 O/                                                            1/1                                          50° C.                                                                          overnight Dowex 50Wx2   Dioxane/H.sub.2 O/                                              (H+)          1/1                                          50° C.                                                                          overnight p.toluene-    Dioxane/H.sub.2 O/                                              sulfonic acid 1/1                                          ______________________________________                                    

Specifically preferred reaction conditions for the preparation ofantibiotic GE 2270 factor A₃ from factor A₂ are the following:

    ______________________________________                                        Temperature                      Polar                                        °C.                                                                              Time      Acid medium  solvent                                      ______________________________________                                        R.T.      15 min    NAOH         Dioxane/H.sub.2 O                                                             1/1                                          R.T.      1 h       0.5M Na.sub.2 CO.sub.3                                                                     Dioxane/H.sub.2 O                                                             1/1                                          R.T.      overnight 0.5M NaHCO.sub.3                                                                           Dioxane/H.sub.2 O                                                             1/1                                          R.T.      overnight 0.5M TEA     Dioxane/H.sub.2 O                                                             1/1                                          80° C.                                                                           1 h       0.5M NaHCO.sub.3                                                                           Dioxane/H.sub.2 O                                                             1/1                                          80° C.                                                                           1 h       0.5M TEA     Dioxane/H.sub.2 O                                                             1/1                                          ______________________________________                                    

Specifically preferred reaction conditions for the preparation ofantibiotic GE 2270 factor A₃ from factor A are the following:

    ______________________________________                                        Temperature                      Polar                                        °C.                                                                             Time      Acid medium   solvent                                      ______________________________________                                        50° C.                                                                          overnight TFA/H.sub.2 O/dioxane                                                         1/4/5                                                      70° C.                                                                          overnight Dowex 50Wx2   Dioxane/H.sub.2 O/                                              (H+)          1/1                                          70° C.                                                                          overnight 0.5N H.sub.2 SO.sub.4                                                                       Dioxane/H.sub.2 O/                                                            1/1                                          ______________________________________                                    

Antibiotic GE 2270 factor H is prepared by antibiotic GE 2270 factor Aby controlled cleavage under reducing conditions.

Preferred reducing conditions are given by alkali metal borohydrides,such as sodium or potassium borohydride, in a compatible polar organicsolvent, such as one of those indicated above. A preferred solvent isaqueous tetrahydrofuran and a preferred borohydride is sodiumborohydride.

The compounds of the invention are recovered at the end of thepreparation process according to known per se techniques which includesextraction with solvents, precipitation by means of precipitatingagents, by changing the pH, and/or by concentration, and chromatographictechniques such as partition chromatography, reverse-phase partitionchromatography, ion-exchange, molecular exclusion chromatography,preparative HPLC and the like.

Physico-chemical Characteristics of Antibiotic GE 2270 Factor A₁

A) ultraviolet absorption spectrum, which is shown in FIG. 1 of theaccompanying drawings, and exhibits the following absorption maxima:

    ______________________________________                                                       Lambda max (nm)                                                ______________________________________                                        0.1M HCl         about 240 (shoulder)                                                          310                                                          0.1M KOH         about 245 (shoulder)                                                          311                                                          Phosphate buffer pH 7.4                                                                        about 245 (shoulder)                                                          310                                                          Methanol         about 215 (shoulder)                                                          about 240 (shoulder)                                                          309                                                          ______________________________________                                    

B) infrared absorption spectrum in nujol mull which is shown in FIG. 2of the accompanying drawings and exhibits the following absorptionmaxima (cm⁻¹): 3700-3000; 3000-2800 (nujol); 1650; 1535; 1505; 1460(nujol); 1375 (nujol); 1310; 1240; 1190; 1165; 1130-1000; 980; 930; 840;805; 750; 720 (nujol); 700;

C) ¹ -NMR spectrum which is shown in FIG. 3 and exhibits the followinggroups of signals at 500 MHz recorded in DMSO-D₆(hexadeuterodimethylsulfoxide) using TMS as the internal standard (0.00ppm); the multiplicity for each signal is also reported below(s=singlet; d=doublet; m=multiplet; dd=doublet of doublets; br s=broadsinglet):

0.84, d; 0.87, d; 1.35, m; 1.91, m; 2.08, m; 2.16, m; 2.46, d; 2.58, s;2.70, dd; 3.38, s; 3.76, m; 3.84, m; 4.26, dd; 4.33, m; 4.89, m; 4.97,s; 5.00, dd; 5.20, dd; 5.22, dd; 5.28 (2 protons), m; 6.01, d; 7.07, s;7.2, s; 7.34, s; 7.22-7.38 (6 protons), m; 8.29, s; 8.39, d; 8.44, m;8.45, d; 8.54, s; 8.60, s; 8.66, d; 9.69, d; 8.99, d.

D) retention-time (R_(t)) of 13.4 min when analyzed by reverse phaseRPLC under the following conditions:

column: Ultrasphere ODS (reverse phase silanized silica gel; 5micrometer) Altex (Beckman) 4.6 mm (i.d.)×250 mm

pre-column: Brownlee Labs RP 18 (octadecylsilane silica gel; 5micrometer)

eluent A: acetonitrile:18 mM NaH₂ PO₄, 70:30 (v/v), adjusted to pH 7.0

eluent B: acetonitrile:18 mM NaH₂ PO₄ 10:90 (v/v), adjusted to pH 7.0

elution mode: linear gradient of eluent A in eluent B from 45% to 70% in20 min

flow rate: 1.8 ml/min

U.V. detector: 254 nm

internal standard: Chloramphenicol (R_(t) =3.6 min)

E) elemental analysis, after the sample has been previously dried atabout 140° C. under inert atmosphere, which indicated the followingcomposition: carbon, hydrogen, nitrogen, sulfur.

F) FAB-MS analysis showing the low mass isotope of the protonatedmolecular ion at m/z 1308 mass units; all the other peaks above 800 m/zmass units in the spectrum, not counting isotope peaks, were lower than20% of the molecular ion, upon analysis with a Kratos MS-50 doublefocusing mass spectrometer under the following experimental conditions:Xe fast atom bombardment at 6 Kv; 0.6 mA discharge current; glycerolmatrix; positive ionization mode

Physico-chemical Characteristics of Antibiotic GE 2270 Factor A₂

A) ultraviolet absorption spectrum, which is shown in FIG. 4 of theaccompanying drawings, and exhibits the following absorption maxima:

    ______________________________________                                                       Lambda max (nm)                                                ______________________________________                                        0.1M HCl         about 245 (shoulder)                                                          309                                                          0.1M KOH         about 245 (shoulder)                                                          309                                                          Phosphate buffer pH 7.4                                                                        about 245 (shoulder)                                                          309                                                          Methanol         about 215 (shoulder)                                                          about 242 (shoulder)                                                          306                                                          ______________________________________                                    

B) infrared absorption spectrum in nujol mull which is shown in FIG. 5of the accompanying drawings and exhibits the following absorptionmaxima (cm⁻¹):

3700-3000; 3000-2800 (nujol); 1725; 1655; 1590-1480; 1460 (nujol); 1410;1375 (nujol); 1335; 1305; 1265-1130; 1090; 1050; 1015; 980; 945; 930;840; 805; 745; 720 (nujol); 700;

C) ¹ H-NMR spectrum which is shown in FIG. 6 and exhibits the followinggroups of signals at 500 MHz recorded in DMSO-D₆hexadeuterodimethylsulfoxide) using TMS as the internal standard (0.00ppm); the multiplicity for each signal is reported between parenthesis(s=singlet; d=doublet; m=multiplet; dd=doublet of doublets; br s=broadsinglet):

0.83, d; 0.86, d; 1.30, m; 1.82, m;; 1.90, m; 2.17, m; 2.46, d; 2.57, s;2.70, dd; 3.37, s; 3.40, m; 3.48, m; 3.77, dd; 4.24, m; 4.28, dd; 4.52,d; 4.53, br s; 4.67, d; 4.96, s; 4.98, dd; 5.19, m; 5.21, m; 5.28, m;6.01, d; 7.34, s; 7.22-7.35, m; 8.26, d; 8.28, s; 8.42, d; 8.45, s;8.59, s; 8.67 (2 protons), d; 8.73, s; 9.00, d;

D) retention-time (R_(t)) of 17.0 min when analyzed by reverse phaseHPLC under the following conditions:

column: Ultrasphere ODS (reverse phase silanized silica gel; 5micrometer) Altex (Beckman) 4.6 mm (i.d.)×250 mm

pre-column: Brownlee Labs RP 18 (octadecylsilane silica gel; 5micrometer)

eluent A: acetonitrile:18 mM NaH₂ PO₄, 70:30 (v/v), adjusted to pH 7.0

eluent B: acetonitrile:18 mM NaH₂ PO₄ 10:90 (v/v), adjusted to pB 7.0

elution mode: linear gradient of eluent A in eluent B from 45% to 70% in20 min

flow rate: 1.8 ml/min

U.V. detector: 254 nm

internal standard: Chloramphenicol (R_(t) =3.6 min)

E) elemental analysis, after the sample has been previously dried atabout 140° C. under inert atmosphere, which indicated the followingcomposition: carbon, hydrogen, nitrogen, sulfur.

F) FAB-MS analysis showing the low mass isotope of the protonatedmolecular ion at m/z 1291 mass units; all the other peaks above 800 M/Zmass units in the spectrum, not counting isotope peaks, were lower than20% of the molecular ion, upon analysis with a Kratos MS-50 doublefocusing mass spectrometer under the following experimental conditions:Xe fast atom bombardment at 6 Kv; 0.6 mA discharge current; glycerolmatrix; positive ionization mode

Physico-chemical Characteristics of Antibiotic GE 2270 Factor A₃

A) ultraviolet absorption spectrum, which is shown in FIG. 7 of theaccompanying drawings, and exhibits the following absorption maxima:

    ______________________________________                                                           Lambda max (nm)                                            ______________________________________                                        0.1M KOH             240 (shoulder)                                                                309                                                                           330 (shoulder)                                           Phosphate buffer pH 7.4                                                                            240 (shoulder)                                                                309                                                                           330 shoulder                                             Methanol             240 (shoulder)                                                                309                                                                           340 (shoulder)                                           ______________________________________                                    

B) infrared absorption spectrum in nujol mull which is shown in FIG. 8of the accompanying drawings and exhibits the following absorptionmaxima (cm⁻¹):

3700-3140; 3110; 3020-2750 (nujol); 1720; 1655; 1590-1520; 1500; 1460(nujol); 1375 (nujol) 1270-1200; 1130-1030; 1020; 980; 930; 840; 805;750; 720 (nujol); 700;

C) ¹ -NMR spectrum which is shown in FIG. 9 and exhibits the followinggroups of signals (in ppm) at 500 MHz recorded in DMSO-D₆hexadeuterodimethylsulfoxide) using TMS as the internal standard (0.00ppm); the number of protons and multiplicity for each signal is alsoreported below (s=singlet; d=doublet; m=multiplet; dd=doublet ofdoublets; br s=broad singlet):

9.02,1H (d); 8.71,1H (d); 8.70,1H (d); 8.65,1H (s); 8.57,1H (s); 8.46,1H(m); 8.38,1H (d); 8.28,1H (d); 8.25,1H (s); 7.38,1H (m); 7.37,1H (s);7.36-7.20,5H (m); 6.05,1H (br s); 5.31,1H (m); 5.27,1H (dd); 5.20,1H(dd); 5.03,1H (d); 4.99,2H (s); 4.32,1H (dd); 3.82,1H (dd); 3.38,3H (s);2.74,1H (dd); 2.60,3H (s); 2.49,3H (d); 2.17,1H (m); 1.35,1H (m);0.88,3H (d); 0.84,3H (d);

D) retention-time (R_(t)) of 7.1 min when analyzed by reverse phase HPLCunder the following conditions:

column: Ultrasphere ODS (reverse phase silanized silica gel; 5micrometer) Altex (Beckman) 4.6 mm (i.d.)×250 mm

pre-column: Brownlee Labs RP 18 (octadecylsilane silica gel; 5micrometer)

eluent A: acetonitrile:18 mM NaH₂ PO₄, 70:30 (v/v), adjusted to pH 7.0

eluent B: acetonitrile:18 mM NaH₂ PO₄ 10:90 (v/v), adjusted to pH 7.0

elution mode: linear gradient of eluent A in eluent B from 45% to 70% in20 min

flow rate: 1.8 ml/min

U.V. detector: 254 nm

internal standard: Chloramphenicol (R_(t) =3.6 min)

E) elemental analysis, after the sample has been previously dried atabout 140° C. under inert atmosphere, which indicated the followingcomposition: carbon 51.27%, hydrogen 4.02%, nitrogen 14.94%, sulfur.

F) FAB-MS analysis showing the low mass isotope of the protonatedmolecular ion at m/z 1125 mass units; all the other peaks above 800 m/zmass units in the spectrum, not counting isotope peaks, were lower than20% of the molecular ion, upon analysis with a Kratos MS-50 doublefocusing mass spectrometer under the following experimental conditions:Xe fast atom bombardment at 6 Kv; 0.6 mA discharge current; glycerolmatrix; positive ionization mode

G) ¹³ C-NMR spectrum which is reported in FIG. 10 of the accompanyingdrawings exhibiting the following groups of signals (ppm) at 125 MHz inDMSO-d₆ TMS as the internal reference (0.00 ppm), 171.2; 169.9; 169.6;168.5; 167.8; 165.7; 164.8; 162.2; 161.4; 161.3; 160.5; 160.4; 153.5;150.4; 150.1; 149.5; 149.1; 147.0; 143.8; 142.1; 141.8; 141.4; 141.0;139.6; 131.8; 128.0 (2 carbons); 127.7; 127.6; 126.9; 126.8 (2 carbons);123.1; 118.7; 116.4; 73.9; 67.4; 58.7; 58.3; 55.5; 48.2; 41.2; 37.7;34.1; 25.9; 18.5; 18.0; 12.0;

H) a specific optical rotation [α]²⁰ _(D) of +182.5 in CHCl₃ +10% CH₃ OH

Physico-chemical Characteristics of Antibiotic GE 2270 Factor H

A) ultraviolet absorption spectrum, which is shown in FIG. 11 of theaccompanying drawings, and exhibits the following absorption maxima:

    ______________________________________                                                       Lambda max (nm)                                                ______________________________________                                        0.1M HCL         248                                                                           303                                                          0.1M KOH         about 245 (shoulder)                                                          310                                                          Phosphate buffer pH 7.4                                                                        about 240 (shoulder)                                                          312                                                          Methanol         about 215 (shoulder)                                                          about 240 (shoulder)                                                          309                                                          ______________________________________                                    

B) infrared absorption spectrum in nujol mull which is shown in FIG. 12of the accompanying drawings and exhibits the following absorptionmaxima (cm⁻¹):

3700-3000; 3000-2800 (nujol); 1655; 1590-1480; 1460 (nujol) 1375(nujol); 1310; 1220; 1190; 1130-1000; 980; 930; 840; 820-680; 720(nujol); 640;

C) ¹ -NMR spectrum which is shown in FIG. 13 and exhibits the followinggroups of signals (ppm) at 500 MHz recorded in DMSO-D₆(hexadeuterodimethylsulfoxide) using TMS as the internal standard (0.00ppm); the multiplicity for each signal is also reported below(s=singlet; d=doublet; m=multiplet; dd=doublet of doublets; br s=broadsinglet):

0.83, d; 0.87 d; 1.30, m; 2.16, m; 2.46, d; 2.58, s; 2.70, dd; 3.38, s;3.52, m; 3.61, m; 3.77, dd; 4.25, m; 4.30, m; 4.35, m; 4.47, m; 4.88, m;4.97, s; 4.99, dd; 5.20, m; 5.23, m; 5.28, m; 6.02, d; 7.36, s;7.22-7.40, m; 8.26, d; 8.28, s; 8.42, d; 8.43, m; 8.47, s; 8.60, s;8.67, d; 9.02, d;

D) retention-time (R_(t)) of 18.0 min when analyzed by reverse phaseHPLC under the following conditions:

column: Ultrasphere ODS (reverse phase silanized silica gel; 5micrometer) Altex (Beckman) 4.6 mm (i.d.)×250 mm

pre-column: Brownlee Labs RP 18 (octadecylsilane silica gel; 5micrometer)

eluent A: acetonitrile:18 mM NaH₂ PO₄, 70:30 (v/v), adjusted to pH 7.0

eluent B: acetonitrile:18 mM NaH₂ PO₄ 10:90 (v/v), adjusted to pH 7.0

elution mode: linear gradient of eluent A in eluent B from 45% to 70% in20 min

flow rate: 1.8 ml/min

U.V. detector: 254 nm

internal standard: Chloramphenicol (R_(t) =3.6 min)

E) elemental analysis, after the sample has been previously dried atabout 140° C. under inert atmosphere, which indicated the followingcomposition: carbon, hydrogen, nitrogen, sulfur.

F) FAB-MS analysis showing the low mass isotope of the protonatedmolecular ion at m/z 1180 mass units; all the other peaks above 800 m/zmass units (not counting isotope peaks) in the spectrum were lower than20% of the molecular ion, upon analysis with a Kratos MS-50 doublefocusing mass spectrometer under the following experimental conditions:Xe fast atom bombardment at 6 Kv; 0.6 mA discharge current; glycerolmatrix; positive ionization mode.

The antimicrobial activity of the compounds of the invention can bedemonstrated by a series of standard tests in vitro.

MIC for Clostridium difficile, Propionibacterium acnes, and Bacteroidesfragilis are determined by agar dilution (inocula 10⁴ /10⁵ CFU/spot).MIC for other organisms are determined by microbroth dilution (inocula10⁴ to 10⁵ CFU/ml). Incubation times are 18-24 h, except for Neisseriagonorrhoeae, Branhamella catarrhalis, Haemophilus influenzae, C.difficile, P. acnes, B. fragilis (48h). All organisms are incubated at37° C. N. gonorrhoeae and H. influenzae are incubated in a 5% CO₂atmosphere, anaerobes in an anaerobic gas mixture. Media used are:Iso-Sensitest broth (Oxoid) (Staphylococci, Streptococcus faecalis,Escherichia coli, Pseudomonas aeruginosa, Proteus vulgaris, Klebsiellapneumoniae); Todd-Hewitt broth (Difco) (other streptococci); MuellerHinton broth (BBL) (Branhamella catarrhalis); GC base broth (Difco)+1%IsoVitalex (BBL) (N. gonorrhoeae); brain heart infusion broth (Difco)+1%Supplement C (Difco) (H. influenzae); AC medium (Difco) (C.perfringens); Wilkins-Chalgren agar (Difco) (other anaerobes) (T. D.Wilkins and S. Chalgren, Antimicrob. Ag. Chemother. 10, 926, 1976).Chlamydia trachomatis was cultivated in microtiter plates oncycloheximide treated McCoy cell monolayers, in Eagle's MEM medium(Gibco) with 10% fetal calf serum, in a 5% CO₂ atmosphere. Inocula wereprepared so as to give 30-60 inclusions per 300× microscope field. After48 h, chlamydial inclusions were stained with fluorescein-labeledmonoclonal antibody to the major outer membrane protein (Syva) andcounted by fluorescence microscopy. The MIC was taken as theconcentration at which inclusions were no longer seen and cellularmorphology was normal.

The minimal inhibitory concentrations (MIC, microgram/ml) for somemicroorganisms are reported below in Table I.

                  TABLE I                                                         ______________________________________                                                                M.I.C.                                                                        (microgram/ml)                                                                Antibiotic                                            Strain                  GE 2270                                               ______________________________________                                                                factor A.sub.1                                        Staph. aureus Tour L165 0.06                                                  Staph. aureus Tour L165 + 30% bovine serum                                                            0.5                                                   Staph. epidermidis L147 ATCC 12228                                                                    0.06                                                  Staph. haemolyticus L602 clin. isolate                                                                ≦0.13                                          Strep. pyogenes C203    >128                                                  Strep. pneumonias UC41  >128                                                  Strep. faecalis ATCC 7080                                                                             0.06                                                  Strep. mitis L796       16                                                    Clostridium perfringens ISS 30543                                                                     0.06                                                  Propionibacterium acnes ATCC 6919                                                                     0.03                                                  Bacteroides fragilis ATCC 23745                                                                       >128                                                  Neisseria gonorrhoeae ISM68/126                                                                       >128                                                  Ureaplasma urealyticum L 1479                                                                         >128                                                  Escherichia coli SKF 12140                                                                            >128                                                  Pseudomonas aeruginosa ATCC 10145                                                                     >128                                                  Proteus vulgaris ATCC 881                                                                             >128                                                  Klebsiella pneumonias L142                                                                            >128                                                  Branhamella catarrhalis ATCC 8176 L76                                                                 >64                                                                           factor A.sub.2                                        Staph. aureus Tour L165 1                                                     Staph. aureus Tour L165 + 30% bovine serum                                                            2                                                     Staph. epidermidis L147 ATCC 12228                                                                    2                                                     Staph. haemolyticus L602 clin. isolate                                                                4                                                     Strep. pyogenes C203    >128                                                  Strep. pneumonias UC41  >128                                                  Strep. faecalis ATCC 7080                                                                             0.5                                                   Strep. mitis L796       8                                                     Clostridium perfringens ISS 30543                                                                     0.06                                                  Propionibacterium acnes ATCC 6919                                                                     0.06                                                  Bacteroides fragilis ATCC 23745                                                                       128                                                   Neisseria gonorrhoeae ISM68/126                                                                       >128                                                  Haemophilus influenzae type b ATCC19418                                                               >128                                                  Ureaplasma urealyticum L 1479                                                                         >128                                                  Escherichia coli SKF 12140                                                                            >128                                                  Pseudomonas aeruginosa ATCC 10145                                                                     >128                                                  Proteus vulgaris ATCC 881                                                                             >128                                                                          factor A.sub.3                                        Staph. aureus Tour L165 0.5                                                   Staph. aureus Tour L165 + 30% bovine serum                                                            2                                                     Staph. epidermidis L147 ATCC 12228                                                                    1                                                     Staph. haemolyticus L602 clin. isolate                                                                2                                                     Strep. pyogenes C203    8                                                     Strep. pneumonias UC41  0.5                                                   Strep. faecalis ATCC 7080                                                                             1                                                     Clostridium perfringens ISS 30543                                                                     ≦0.03                                          Propionibacterium acnes ATCC 6919                                                                     ≦0.03                                          Bacteroides fragilis ATCC 23745                                                                       8                                                     Neisseria gonorrhoeae ISM68/126                                                                       8                                                     Haemophilus influenzae type b ATCC19418                                                               4                                                     Ureaplasma urealyticum L 1479                                                                         >128                                                  Escherichia coli SKF 12140                                                                            >128                                                  Pseudomonas aeruginosa ATCC 10145                                                                     >128                                                  Proteus vulgaris ATCC 881                                                                             >128                                                  Branhamella catarrhalis ATCC 8176 L 76                                                                0.5                                                   Chlamydia trachomatis(oculo-genital clin.isol.)                                                       128                                                                           factor H                                              Staph. aureus Tour L165 0.06                                                  Staph. aureus Tour L165 + 30% bovine serum                                                            0.25                                                  Staph. epidermidis L147 ATCC 12228                                                                    0.25                                                  Staph. haemolyticus L602 clin. isolate                                                                0.5                                                   Strep. pyogenes C203    4                                                     Strep. pneumoniae UC41  1                                                     Strep. faecalis ATCC 7080                                                                             0.06                                                  Strep. mitis L796       0.5                                                   Clostridium perfringens ISS 30543                                                                     0.03                                                  Propionibacterium acnes ATCC 6919                                                                     0.25                                                  Bacteroides fragilis ATCC 23745                                                                       >64                                                   Neisseria gonorrhoeae ISM68/126                                                                       >128                                                  Haemophilus influenzae type b ATCC 19418                                                              >128                                                  Ureaplasma urealyticum L 1479                                                                         32                                                    Escherichia coli SKF 12140                                                                            >128                                                  Pseudomonas aeruginosa ATCC 10145                                                                     >128                                                  Proteus vulgaris ATCC 881                                                                             >128                                                  Klebsiella pneumoniae L142                                                                            >128                                                  ______________________________________                                    

The antimicrobial activity of the compounds of the invention isconfirmed also in experimental septicemia in mice.

Groups of five CD1 mice of both sexes (Charles River, average weight18-22 g) were infected intraperitoneally with Staphylococcus aureus ATCC19636. The bacterial challenge (10⁶ cells/mouse) was given suspended in0.5 ml of 5% bacteriological mucin (Difco). The test compounds wereadministered intraveneously once immediately after infection in asterile aqueous solution containing 5% dimethylformamide and 10%Chremophor® EL (polyethoxylated castor oil).

The ED₅₀ was calculated on the seventh day from the percentage ofsurviving animals at each dose by the Spearman and Kaerber method; itsvalue resulted 15.4 mg/kg for antibiotic GE 2270 factor A₃ and 3.2 mg/kgfor antibiotic GE 2270 factor A₁.

In a similar experiment, but using groups of eight mice andadministering a single dose level of test compound (20 mg/kg), the ratiosurvivors/treated at the seventh day was 1/8 in the case of antibioticGE 2270 factor A₂ and 6/8 in the case of antibiotic GE 2270 factor H.The test compounds were given i.v. as a milky suspension in 10%dimethylformamide in 5% aqueous glucose.

In view of their properties, the compounds of the invention can be usedas active ingredients in the preparation of medicaments for human oranimal treatment.

In particular, antibiotic GE 2270 factor A₁, A₂, A₃ and H areantimicrobial agents mainly active against gram positive bacteria andgram positive as well as gram negative anaerobes.

The main therapeutic indication of the antibiotic substances of theinvention is thus in the treatment of infections related to the presenceof microorganisms susceptible to them.

The term "treatment" is intended to encompass also prophylaxis, therapyand cure.

The patient receiving this treatment is any animal in need, includingprimates, in particular humans, and other mammals such as equines,cattle, swine and sheep; and poultry and pets in general.

The compounds of the invention can be administered as such or inadmixture with pharmaceutically acceptable carriers and can also beadministered in conjunction with other antimicrobial agents. Conjunctivetherapy, thus includes sequential, simultaneous and separateadministration of the active compounds in a way that the therapeuticaleffects of the first administered one is not entirely disappeared whenthe subsequent is administered.

A preferred pharmaceutical formulation is represented by a formulationsuitable for a topical application on an intact or damaged skin ormucous membrane. Examples of such formulations are powders, ointments,creams and lotions. The excipients in these formulations are the usualpharmaceutically acceptable vehicles such oleaginous ointment bases(e.g. cetyl esters wax, oleic acid, olive oil, paraffin, spermaceti,starch glycerite); absorbent ointment bases (e.g. anhydrous lanolin,hydrophilic petrolatum), emulsion ointment bases (e.g. cetyl alcohol,glyceryl monostearate, lanolin, stearic acid), water-soluble ointmentbases (e.g. glycol ethers and their derivatives which includepolyethylene glycols, poly(oxy-1,2-ethanediyl)-alpha-hydro-omega-hydroxy-octadecanoate, polysorbates, andpolyethylene glycols mono-stearates).

These formulations may contain other known excipients, such aspreservatives and are prepared as known in the art and reported inreference handbooks such as Remington's Pharmaceutical Sciences,Seventeenth edition, 1985, Mack Publishing Co.

The compounds of the invention can also be formulated into formulationsuitable for parenteral administration according to procedures known perse in the art. For instance, a compound of the invention is formulatedwith polypropylene glycol or dimethylacetamide and a surface-activeagent such as polyoxyethylene sorbitan mono-oleate or polyethoxylatedcastor oil.

A preferred formulation for parenteral administration includes thefollowing excipients: Cremophor® EL (polyoxyl 35 castor oil USP/NF) 20%,propylene glycol 5-10%.

Preferably, this formulation is used for i.v. administration in thetreatment of any infection involving a microorganism susceptible to anantibiotic of the invention.

In the treatment of pseudomembranous colitis or other diseasesattributable to the presence of anaerobes in the gastrointestinal tract,an effective dose of the compounds of the invention may be administeredorally in suitable pharmaceutical form such as a capsule or an aqueoussuspension.

The dosage of the active ingredient depends on many factors whichinclude type, age and conditions of the patient, specific activeingredient and formulation selected for administration, administrationschedule, etc.

In general, effective antimicrobial dosages are employed per single unitdosage form. Repeated applications of these dosage forms, e.g. from 2 to6 times a day, are in general preferred. An effective dosage may be ingeneral in the range 0.5-50 mg/kg body weight/day.

A preferred topic preparation is an ointment containing from 1% to 10%of a compound of the present invention.

Anyway, the prescribing physician will be able to determine the optimaldosage for a given patient in a given situation.

Besides their use as medicaments in human and veterinary therapy, thecompounds of the invention can also be used as animal growth promoters.

For this purpose, a compound of the invention is administered orally ina suitable feed. The exact concentration employed is that which isrequired to provide for the active agent in a growth promotant effectiveamount when normal amounts of feed are consumed.

The addition of the active compound of the invention to animal feed ispreferably accomplished by preparing an appropriate feed premixcontaining the active compound in an effective amount and incorporatingthe premix into the complete ration.

Alternatively, an intermediate concentrate or feed supplement containingthe active ingredient can be blended into the feed. The way in whichsuch feed premixes and complete rations can be prepared and administeredare described in reference books (such as "Applied Animal Nutrition", W.H. Freedman and CO., S. Francisco, USA, 1969 or "Livestock Feeds andFeeding" O and B books, Corvallis, Oregon, USA, 1977).

The following examples further illustrate the invention and should notbe interpreted as limiting it in any way.

EXAMPLE 1 Preparation of Antibiotic GE2270 Factor A₁

1.1 1M Hydrochloric acid (3.3 ml) is added to a solution of 150 mg ofantibiotic GE 2270 factor A in 16 ml of 95% ethanol. The mixture is keptat room temperature for 5 min, diluted with 0.1M sodium phosphate bufferpH 7.5 and then adjusted to pH 7.5 with sodium hydroxide. Afterconcentration to aqueous phase under reduced pressure, this mixture isextracted twice with ethyl acetate. Then the organic phase (whichcontains mainly antibiotic GE 2270 factor A₁) is concentrated to a solidresidue that is solubilized in 5 ml of tetrahydrofuran and then dilutedwith water to the solubility limit. This solution is purified in 10subsequent HPLC runs using a column (250×20 mm) of Nucleosil® C 18 (5micromole) reverse phase silica gel packed by Stacroma^(R) eluting witha linear gradient from 64% to 93% of phase B in phase A, in 20 min, at aflow rate of about 15 ml/min. In this system, phase A is a 90:10 (v/v)mixture of 18 mM sodium phosphate pH 7.2 and acetonitrile, while phase Bis a 40:60 (v/v) mixture of 18 mM sodium phosphate pH 7.2 andacetonitrile. Fractions are collected and UV monitored at 330 nm; thosefractions which contain substantial amounts of antibiotic GE 2270 factorA₁, which correspond to the peaks of the UV plot, are pooled andconcentrated under reduced pressure to aqueous phase which is extractedtwice with ethyl acetate. This organic layer is then washed withdistilled water to remove the residual inorganic salts and concentratedto a solid residue that is then dissolved in tetrahydrofuran andre-precipitated with petroleum ether, thus giving pure antibiotic GE2270 factor A₁ (64 mg).

1.2 Antibiotic GE 2270 factor A₁ is the main reaction product ofantibiotic GE 2270 factor A when this antibiotic is incubated for 15 minat room temperature in a 5/4/1 (v/v) mixture of diethylenedioxide, waterand trifluoroacetic acid.

Antibiotic GE 2270 factor A₁ is also obtained when antibiotic GE 2270factor A is incubated in 0.5N sulfuric acid or 0.25M phosphoric acid ina 1/1 (v/v) mixture of diethylenedioxide and water at room temperature.After dilution with cold water and neutralization, the mixture isextracted with ethyl acetate, the organic phase is concentrated and asolid precipitated by adding petroleum ether.

Pure antibiotic GE 2270 factor A₁ is obtained by preparative HPLC asdescribed in Example 1.1.

1.3 Antibiotic GE 2270 factor A₁ is the main reaction product ofantibiotic GE 2270 factor A when this antibiotic is incubated overnightat 50° C. in 0.5M ammonium chloride in a 1:1 (v/v) mixture ofdiethylenedioxide/water. After dilution with cold water andneutralization, the mixture is extracted with ethyl acetate, the organicphase is concentrated and a solid precipitated by adding petroleumether.

Pure antibiotic GE 2270 factor A₁ is obtained by preparative HPLC asdescribed in Example 1.1.

EXAMPLE 2 Preparation of Antibiotic GE2270 Factor A₂

2.1 Antibiotic GE 2270 factor A (86 mg) is dissolved in 17 ml of 95%ethanol and 1.7 ml of acetic acid. After incubation at 60° C. for 24 h,the resulting solution is diluted with 0.1M sodium phosphate buffer pH7.5 (100 ml) and adjusted to pH 7.5 with 1M sodium hydroxide. Ethanol isremoved by evaporation under reduced pressure and the aqueous residue isextracted twice with ethyl acetate (100 ml). The organic phase isconcentrated under reduced pressure to obtain a solid residue which issolubilized with tetrahydrofuran and then precipitated by addingpetroleum ether. Antibiotic GE 2270 factor A₂ (62 mg) is obtained withminor amounts of antibiotic GE 2270 factors A and A₁. Pure antibiotic GE2270 factor A₂ is obtained by preparative HPLC as follows:

10 Mg of the above crude product is solubilized intetrahydrofuran/diluted to the solubility limit with water and theninjected into a HPLC system with a column (250×20 mm) packed withNucleosil^(R) C₁₈ (5 micrometer) reverse phase silica gel byStacroma^(R), eluting with a linear gradient from 64% to 93% of phase Bin phase A, in 20 min, at a flow rate of about 15 ml/min. In thissystem, phase A is a 90:10 (v/v) mixture of 18 mM sodium phosphate pH7.2 and acetonitrile, while phase B is a 40:60 (v/v) mixture of 18 mMsodium phosphate pH 7.2 and acetonitrile. Fractions of five consecutiveruns are collected and UV monitored at 330 nm. Fractions which containsubstantial amounts of antibiotic GE 2270 factor A₂, which correspond tothe major peaks of the UV elution profile, are pooled and concentratedunder reduced pressure to an aqueous phase which is extracted twice withethyl acetate. This organic layer is then washed with distilled water toremove the residual inorganic salts and concentrated to precipitate asolid residue that is then dissolved in tetrahydrofuran andreprecipitated with petroleum ether, to obtain pure antibiotic GE 2270factor A₂ (45 mg).

2.2 Antibiotic GE 2270 factor A₂ is the main reaction product ofantibiotic GE 2270 factor A when this antibiotic is incubated overnightat 80° C. in a 1/1 (v/v) mixture of acetic acid and water. Afterdilution with water and neutralization, antibiotic GE 2270 factor A₂ isextracted with ethyl acetate, concentrated under reduced pressure andprecipitated by adding petroleum ether.

Pure antibiotic GE 2270 factor A₂ derivative is obtained by preparativeHPLC as described in Example 2.1.

2.3 Antibiotic GE 2270 factor A₂ is the main reaction product ofantibiotic GE 2270 factor A when this antibiotic is incubated overnightat 50° C. in a 5/4/1 mixture (v/v) of diethylenedioxide, water andtrifluoroacetic acid. Alternatively, the reaction may be conducted inthe presence of inorganic acids such as 0.5N sulfuric acid or 0.25Mphosphoric acid in a 1/1 (v/v) mixture of diethylenedioxide and water.After dilution with water and neutralization, antibiotic GE 2270 factorA₂ is extracted with ethyl acetate, concentrated under reduced pressureand precipitated by adding petroleum ether.

Pure antibiotic GE 2270 factor A₂ can be obtained by preparative HPLC asdescribed in Example 2.1.

2.4 Antibiotic GE 2270 factor A₂ is the main reaction product ofantibiotic GE 2270 factor A when this antibiotic, solubilized in a 1/1(v/v) mixture of diethylenedioxide and water, is incubated overnight at50° C. in the presence of an acidic cation exchange resin such asDowex^(R) 50Wx2 in the acid ionic form. Then the resin is removed fromthe reaction mixture and the solution is diluted with cold water andneutralized. Antibiotic GE 2270 factor A₂ is extracted with ethylacetate, concentrated under reduced pressure and precipitated by addingpetroleum ether.

Pure antibiotic GE 2270 factor A₂ can be obtained by preparative HPLC asdescribed in Example 2.1.

2.5 Antibiotic GE 2270 factor A₂ is also obtained from antibiotic GE2270 factor A₁ when this antibiotic is treated in the conditionsdescribed for GE 2270 factor A in Examples 2.1, 2.2, 2.3 or 2.4.

Generally, a molar yield of 60-85% is obtained in this reaction.

EXAMPLE 3 Preparation of Antibiotic GE2270 Factor A₃

3.1 Antibiotic GE 2270 factor A₂ is incubated for 1 h at roomtemperature in 0.5M sodium carbonate. The reaction mixture is thendiluted with cold water and brought to pH 6.5 with hydrochloric acid.The neutralized solution contains antibiotic GE 2270 factor A₃ as themain reaction product. This antibiotic is extracted from the aqueousphase with ethyl acetate and then is precipitated from the concentratedorganic phase by adding petroleum ether.

Pure antibiotic GE 2270 factor A₃ is obtained by column chromatographyas described below.

3.2 1.5 Grams of crude GE 2270 A₃ is dissolved in 60 ml of a 1/1 (v/v)mixture of methanol and dichloromethane and adsorbed on silica gel(75-230 mesh) by evaporation of the solvents under reduced pressure. Thesolid residue is then put on the top of a silica gel (75-230 mesh)column (bed height 40 cm) equilibrated with dichloromethane. The columnis then eluted with mixtures of methanol in dichloromethane in theorder: 1) 2% methanol (450 ml); 2) 5% methanol (500 ml); 3) 10% methanol(600 ml); 4) 15% methanol (500 ml); 5) 20% methanol (500 ml); 6) 30%methanol (250 ml).

Fractions are collected and monitored by TLC and a microbiological assayon B. subtilis ATCC 6633. Antibiotic GE 2270 factor A₃ is normallypresent in the eluates which contain about 15-20% methanol.

The fractions containing the desired product are pooled and concentratedunder reduced pressure. Upon addition of petroleum ether to the residue,antibiotic GE 2270 factor A₃ precipitates (854 mg of pure product).

3.3 Antibiotic GE 2270 factor A solubilized in a 1/1 (v/v) mixture ofdiethylenedioxide and water is incubated for 40 h in the presence of acation exchange resin (Dowex^(R) 50Wx2) in the H+ form. The resin isthen removed and the reaction solution is diluted with cold water andbrought to pH 6.5 with aqueous sodium hydroxide. This mixture isextracted with ethyl acetate and precipitated from the concentratedorganic phase by adding petroleum ether. Antibiotic GE 2270 factor A₃ isthen isolated and purified by column chromatography as described inExample 3.2. (Global molar yield: 15%).

EXAMPLE 4 Preparation of Antibiotic GE2270 Factor H

4.1 500 Mg of sodium borohydride and 250 mg of antibiotic GE 2270 factorA are dissolved in 100 ml of a 1/1 (v/v) mixture of tetrahydrofuran andwater. After three days at room temperature, the solution is brought toabout pH 7 with a 1M NaH₂ PO₄ and then concentrated under reducedpressure. The water is extracted twice with ethyl acetate and theorganic phase is concentrated to dryness. The solid residue, thatcontains mainly antibiotic GE 2270 factor H, is dissolved intetrahydrofuran and then precipitated by adding petroleum ether thusyielding 213 mg of a white precipitate which is purified by preparativeHPLC as follows:

10 Mg of crude reaction product is solubilized in tetrahydrofuran,diluted to the solubility limit with water and then injected into a HPLCcolumn (250×20 mm) of Nucleosil^(R) C₁₈ (5 micrometer) reverse phasesilica gel packed by Stacroma eluting with a linear gradient from 64% to93% of phase B in phase A, in 20 min, at a flow rate of about 15 ml/min.Then elution is continued for 5 min with phase B alone. In this system,phase A is a 90:10 (v/v) mixture of 18 mM sodium phosphate pH 7.2 andacetonitrile, while phase B is a 40:60 (v/v) mixture of 18 mM sodiumphosphate pH 7.2 and acetonitrile. Fractions of ten consecutive runs arecollected and UV monitored at 330 nm; those fractions which containsubstantial amounts of antibiotic GE 2270 factor H, which correspond tothe peaks of the UV elution profile, are pooled and concentrated underreduced pressure to an aqueous phase which is extracted twice with ethylacetate. This organic layer is then washed with distilled water toremove the residual inorganic salts and concentrated to precipitate asolid residue. This is then dissolved in tetrahydrofuran andre-precipitated with petroleum ether, to obtain pure antibiotic GE 2270factor H (55 mg).

Preparation of the Starting Materials

The starting materials are prepared by following the procedures outlinedin European Patent Application Publication No. 359062.

BRIEF DESCRIPTION OF THE DRAWINGS

The symbols used in the attached U.V. spectra have the followingmeanings:

refers to the assay in 0.1N HCl

refers to the assay in 0.1N KOH

refers to the assay in methanol

refers to assay in phosphate buffer pH 7.4

We claim:
 1. A process for preparing antibiotic GE 2270 factor H and theacceptable salts thereof, which has the following features in theunsalified form:Physico-chemical characteristics of antibiotic GE 2270factor H A) ultraviolet absorption spectrum which exhibits the followingabsorption maxima:

    ______________________________________                                                       Lambda max (nm)                                                ______________________________________                                        0.1M HCL         248                                                                           303                                                          0.1M KOH         about 245 (shoulder)                                                          310                                                          Phosphate buffer pH 7.4                                                                        about 240 (shoulder)                                                          312                                                          Methanol         about 215 (shoulder)                                                          about 240 (shoulder)                                                          309                                                          ______________________________________                                    

B) infrared absorption spectrum in nujol mull which exhibits thefollowing absorption maxima (cm⁻¹): 3700-3000; 3000-2800 (nujol); 1655;1590-1480; 1460 (nujol) 1375 (nujol); 1310; 1220; 1190; 1130-1000; 980;930; 840; 820-680; 720 (nujol); 640; C) ¹ H-NMR spectrum which exhibitsthe following groups of signals (ppm) at 500 MHz recorded in DMSO-D₆(hexadeuterodimethylsulfoxide) using TMS as the internal standard (0.00ppm); the multiplicity for each signal is also reported below(s=singlet; d=doublet; m=multiplet; dd=doublet of doublets; br s=broadsinglet): 0.83, d; 0.87 d; 1.30, m; 2.16, m; 2.46, d; 2.58, s; 2.70, dd;3.38, s; 3.52, m; 3.61, m; 3.77, dd; 4.25, m; 4.30, m; 4.35, m; 4.47, m;4.88, m; 4.97, s; 4.99, dd; 5.20, m; 5.23, m; 5.28, m; 6.02, d; 7.36, s;7.22-7.40, m; 8.26, d; 8.28, s; 8.42, d; 8.43, m; 8.47, s; 8.60, s;8.67, d; 9.02, d; D) retention-time (R_(t)) of 18.0 min when analyzed byreverse phase HPLC under the following conditions: column: UltrasphereODS (reverse phase silanized silica gel; 5 micrometer) Altex (Beckman)4.6 mm (i.d.)×250 mm pre-column: Brownlee Labs RP 18 (octadecylsilanesilica gel; 5 micrometer) eluent A: acetonitrile:18 mM NaH₂ PO₄, 70:30(v/v), adjusted to pH 7.0 eluent B: acetonitrile:18 mM NaH₂ PO₄ 10:90(v/v), adjusted to pH 7.0 elution mode: linear gradient of eluent A ineluent B from 45% to 70% in 20 min flow rate: 1.8 ml/min U.V. detector:254 nm internal standard: Chloramphenicol (R_(t) =3.6 min) E) elementalanalysis, after the sample has been previously dried at about 140° C.under inert atmosphere, which indicated the following composition:carbon, hydrogen, nitrogen, sulfur, F) FAB-MS analysis showing the lowmass isotope of the protonated molecular ion at m/z 1180 mas units; allthe other peaks above 800 m/z mass units (not counting isotope peaks) inthe spectrum were lower than 20% of the molecular ion, upon analysiswith a Kratos MS-50 double focusing mass spectrometer under thefollowing experimental conditions: Xe fast atom bombardment at 6 Kv; 0.6mA discharge current; glycerol matrix; positive ionization mode,whichcomprises: 1) subjecting antibiotic GE 2270 factor A to a reductivecleavage 2) with an alkali metal borohydride; 3) in a polar organicsolvent selected from the group consisting of lower alkanols, phenylsubstituted lower alkanols, lower alkyl carboxamides, lower alkylsulfoxamides, lower alkyl phosphoramides, lower alkyl sulfoxides, loweralkyl sulfones and cyclic oxygen containing aliphatic solvents; 4)monitoring said reaction for the appearance of said Antiotic GE 2270factor; 5) allowing said reaction to continue for a period of timesufficient to obtain antibiotic GE 2270 factor H and recovering saidfactor from the reaction medium.
 2. A process for preparing anantibiotic substance selected from antibiotic GE 2270 factor A₁ or anacceptable salt thereof, which have the following features in theunsalified form:Physico-chemical Characteristics of Antibiotic GE 2270Factor A₁ A) ultraviolet absorption spectrum which exhibits thefollowing absorption maxima:

    ______________________________________                                                       Lambda max (nm)                                                ______________________________________                                        0.1M HCl         about 240 (shoulder)                                                          310                                                          0.1M KOH         about 245 (shoulder)                                                          311                                                          Phosphate buffer pH 7.4                                                                        about 245 (shoulder)                                                          310                                                          Methanol         about 215 (shoulder)                                                          about 240 (shoulder)                                                          309                                                          ______________________________________                                    

B) infrared absorption spectrum in nujol mull which exhibits thefollowing absorption maxima (cm⁻¹): 3700-3000; 3000-2800 (nujol); 1650;1535; 1505; 1460 (nujol); 1375 (nujol); 1310; 1240; 1190; 1165;1130-1000; 980; 930; 840; 805; 750; 720 (nujol); 700; C) ¹ H-NMRspectrum which exhibits the following groups of signals at 500 MHzrecorded in DMSO-D₆ (hexadeuterodimethylsulfoxide) using TMS as theinternal standard (0.00 ppm); the multiplicity for each signal is alsoreported below (s=singlet; d=doublet; m=multiplet; dd=doublet ofdoublets; br s=broad singlet): 0.84, d; 0.87, d; 1.35, m; 1.91, m; 2.08,m; 2.16, m; 2.46, d; 2.58, s; 2.70, dd; 3.38, s; 3.76, m; 3.84, m; 4.26,dd; 4.33, m; 4.89, m; 4.97, s; 5.00, dd; 5.20, dd; 5.22, dd; 5.28 (2protons), m; 6.01, d; 7.07, s; 7.2, s; 7.34, s; 7.22-7.38 (6 protons),m; 8.29, s; 8.39, d; 8.44, m; 8.45, d; 8.54, s; 8.60, s; 8.66, d; 9.69,d; 8.99, d D) retention-time (R_(t)) of 13.4 min when analyzed byreverse phase HPLC under the following conditions: column: UltrasphereODS (reverse phase silanized silica gel; 5 micrometer) Altex (Beckman)4.6 mm (i.d.)×250 mm pre-column: Brownlee Labs RP 18 (octadecylsilanesilica gel; 5 micrometer) eluent A: acetonitrile:18 mM NaH₂ PO₄, 70:30(v/v), adjusted to pH 7.0 eluent B: acetonitrile:18 mM NaH₂ PO₄ 10:90(v/v), adjusted to pH 7.0 elution mode: linear gradient of eluent A ineluent B from 45% to 70% in 20 min flow rate: 1.8 ml/min U.V. detector:254 nm internal standard: Chloramphenicol (R_(t) =3.6 min) E) elementalanalysis, after the sample has been previously dried at about 140° C.under inert atmosphere, which indicated the following composition:carbon, hydrogen, nitrogen, sulfur, F) FAB-MS analysis showing The lowmass isotope of the protonated molecular ion at m/z 1308 mass units; allthe other peaks above 800 m/z mass units in the spectrum, not countingisotope peaks, were lower than 20% of the molecular ion, upon analysiswith a Kratos MS-50 double focusing mass spectrometer under thefollowing experimental conditions: Xe fast atom bombardment at 6 Kv; 0.6mA discharge current; glycerol matrix; positive ionization modewhichcomprises subjecting antibiotic GE 2270 factor A to a hydrolysisreaction: 1) with an aqueous solution or suspension of a mineral acid inwhich said mineral acid is selected from the group consisting ofhydrogen chloride, hydrogen bromide, hydrogen iodide, sulfuric acid andphosphoric acid, or with an aqueous solution or suspension an organicacid in which said organic acid is selected from the group consisting ofC₁ -C₆) aliphatic acids, halogenated(C₂ -C₅) aliphatic acids, arylicacids, C₂ -C₆)alkyl sulfonic acids, and arylic sulfonic acids; 2) in thepresence of a polar organic solvent selected from the group consistingof lower alkanols, phenyl substituted lower alkanols, lower alkylcarboxamides, lower alkyl sulfoxamides, lower alkyl phosphoramides,lower alkyl sulfoxides, lower alkyl sulfones and cyclic oxygencontaining aliphatic solvents; 3) at a temperature ranging from -10° C.to 50° C.; 4) for a period of time ranging from between 5 minutes and 16hours, and; 5) monitoring the reaction for the appearance of saidantibiotic GE 2270 factor A1 and recovering said antibiotic GE 2770factor A₁ from the reaction medium.
 3. A process for reducint antibioticGE 2270 factor A2 or an acceptable salt thereof; which has the followingfeatures in the unsalified form:Physico-chemical characteristics ofantibiotic GE 2270 factor A₂ A) ultraviolet absorption spectrum whichexhibits the following absorption maxima:

    ______________________________________                                                       Lambda max (nm)                                                ______________________________________                                        0.1M HCl         about 245 (shoulder)                                                          309                                                          0.1M KOH         about 245 (shoulder)                                                          309                                                          Phosphate buffer pH 7.4                                                                        about 245 (shoulder)                                                          309                                                          Methanol         about 215 (shoulder)                                                          about 242 (shoulder)                                                          306                                                          ______________________________________                                    

B) infrared absorption spectrum in nujol mull which exhibits thefollowing absorption maxima (cm⁻¹): 370- 3000; 3000-2800 (nujol); 1725;1655; 1590-1480; 1460 (nujol); 1410; 1375 (nujol); 1335; 1305;1265-1130; 1090; 1050; 1015; 980; 945; 930; 840; 805; 745; 720 (nujol);700; C) ¹ H-NMR spectrum which exhibits the following groups of signalsat 500 MHz recorded in DMSO-D₆ hexadeuterodimethylsulfoxide) using TMSas the internal standard (0.00 ppm); the multiplicity for each signal isreported between parenthesis (s=singlet; d=doublet; m=multiplet;dd=doublet of doublets; br s=broad singlet): 0.83, d; 0.86, d; 1.30, m;1.82, m;; 1.90, m; 2.17, m; 2.46, d; 2.57, s; 2.70, dd; 3.37, s; 3.40,m; 3.48, m; 3.77, dd; 4.24, m; 4.28, dd; 4.52, d; 4.53, br s; 4.67, d;4.96, s; 4.98, dd; 5.19, m; 5.21, m; 5.28, m; 6.01, d; 7.34, s;7.22-7.35, m; 8.26, d; 8.28, s; 8.42, d; 8.45, s; 8.59, s; 8.67 (2protons), d; 8.73, s; 9.00, d; D) retention-time (R_(t)) of 17.0 minwhen analyzed by reverse phase HPLC under the following conditions:column: Ultrasphere ODS (reverse phase silanized silica gel; 5micrometer) Altex (Beckman) 4.6 mm (i.d.)×250 mm pre-column: BrownleeLabs RP 18 (octadecylsilane silica gel; 5 micrometer) eluent A:acetonitrile:18 mM NaH₂ PO₄, 70:30 (v/v), adjusted to pH 7.0 eluent B:acetonitrile:18 mM NaH₂ PO₄ 10:90 (v/v), adjusted to pH 7.0 elutionmode: linear gradient of eluent A in eluent B from 45% to 70% in 20 minflow rate: 1.8 ml/min U.V. detector: 254 nm internal standard:Chloramphenicol (R_(t) =3.6 min) E) elemental analysis, after the samplehas been previously dried at about 140° C. under inert atmosphere, whichindicated the following composition: carbon, hydrogen, nitrogen, sulfur,F) FAB-MS analysis showing the low mass isotope of the protonatedmolecular ion at m/z 1291 mass units; all the other peaks above 800 M/Zmass units in the spectrum, not counting isotope peaks, were lower than20% of the molecular ion, upon analysis with a Kratos MS-50 doublefocusing mass spectrometer under the following experimental conditions:Xe fast atom bombardment at 6 Kv; 0.6 mA discharge current; glycerolmatrix; positive ionization modewhich comprises subjecting antibiotic GE2270 factor A or factor A₁ to a hydrolysis reaction: 1) with an aqueoussolution or suspension of a mineral acid in which said mineral acid isselected from the group consisting of hydrogen chloride, hydrogenbromider hydrogen iodide, sulfuric acid and phosphoric acid, or with anaqueous solution or suspension an organic acid in which said organicacid is selected from the group consisting of (C₁ -C₆) aliphatic acids,halogenated(C₂ -C₅) aliphatic acids, arylic acids, (C₂ -C₆)alkylsulfonicacids, and arylic sulfonic acids; 2) in the presence of apolar organicsolvent selected from the group consisting of lower alkanols, phenylsubstituted lower alkanols, lower alkyl carboxamides, lower alkylsulfoxamides, lower alkyl phosphoramides, lower alkyl sulfoxides, loweralkyl sulfones and cyclic oxygen containing aliphatic solvents; 3) at atemperature ranging from 40° C. to 90° C.; 4) for a period of timeranging from 12 to 24 hours, and; 5) monitoring the reaction for theappearance of said antibiotic GE 2270 factor A₂ and recovering saidsantibiotic GE 2270 factor A₂ from the reaction medium.
 4. A process forproducing antibiotic GE 2270 factor A3 or an acceptable salt thereof,which has the following feature in the unsalified form:Physico-chemicalcharacteristics of antibiotic GE 2270 factor A₃ A) ultravioletabsorption spectrum which exhibits the following absorption maxima:

    ______________________________________                                                       Lambda max (nm)                                                ______________________________________                                        O.1 M KOH        240        (shoulder)                                                         309                                                                           330        (shoulder)                                        Phosphate buffer pH 7.4                                                                        240        (shoulder)                                                         309                                                                           330        shoulder                                          Methanol         240        (shoulder)                                                         309                                                                           340        (shoulder)                                        ______________________________________                                    

B) infrared absorption spectrum in nujol mull which exhibits thefollowing absorption maxima (cm⁻¹): 3700-3140; 3110; 3020-2750 (nujol);1720; 1655; 1590-1520; 1500; 1460 (nujol); 1375 (nujol) 1270-1200;1130-1030; 1020; 980; 930; 840; 805; 750; 720 (nujol); 700; C) ¹ H-NMRspectrum which exhibits the following groups of signals (in ppm) at 500MHz recorded in DMSO-D₆ hexadeuterodimethylsulfoxide) using TMS as theinternal standard (0.00 ppm); the number of protons and multiplicity foreach signal is also reported below (s=singlet; d=doublet; m=multiplet;dd=doublet of doublets; br s=broad singlet):
 9. 02,1H (d); 8.7L,1H (d);8.70,1H (d); 8.65,1H (s); 8.57,1H (s); 8.46,1H (m); 8.38,1H (d); 8.28,1H(d); 8.25,1H (s); 7.38,1H (m); 7.37,1H (s); 7.36-7.20,5H (m); 6.05,1H(br s); 5.31,1H (m); 5.27,1H (dd); 5.20,1H (dd); 5.03,1H (d); 4.99,2H(s); 4.32,1H (dd); 3.82,1H (dd); 3.38,3H (s); 2.74,1H (dd); 2.60,3H (s);2.49,3H (d); 2.17,1H (m); 1.35,1H (m); 0.88,3H (d); 0.84,3H (d);D)retention-time (R_(t)) of 7.1 min when analyzed by reverse phase HPLCunder the following conditions: column: Ultrasphere ODS (reverse phasesilanized silica gel; 5 micrometer) Altex (Beckman) 4.6 mm (i.d.)×250 mmpre-column: Brownlee Labs RP 18 (octadecylsilane silica gel; 5micrometer) eluent A: acetonitrile:18 mM NaH₂ PO₄, 70:30 (v/v), adjustedto pH 7.0 eluent B: acetonitrile:18 mM NaH₂ PO₄ 10:90 (v/v), adjusted topH 7.0 elution mode: linear gradient of eluent A in eluent B from 45% to70% in 20 min flow rate: 1.8 ml/min U.V. detector: 254 nm internalstandard: Chloramphenicol (R_(t) =3.6 min) E) elemental analysis, afterthe sample has been previously dried at about 140° C. under inertatmosphere, which indicated the following composition: carbon 51.27%,hydrogen 4.02%, nitrogen 14.94%, sulfur, F) FAB-MS analysis snowing thelow mass isotope of the protonated molecular ion at m/z 1125 mass units;all the other peaks above 800 m/z mass units in the spectrum, notcounting isotope peaks, were lower than 20% of the molecular ion, uponanalysis with a Kratos MS-50 double focusing mass spectrometer under thefollowing experimental conditions: Xe fast atom bombardment at 6 Kv; 0.6mA discharge current; glycerol matrix; positive ionization mode G) ¹³C-NMR spectrum exhibiting the following groups of signals (ppm) at 125MHz in DMSO-d₆ TMS as the internal reference (0.00 ppm), 171.2; 169.9;169.6; 168.5; 167.8; 165.7; 164.8; 162.2; 161.4; 161.3; 160.5; 160.4;153.5; 150.4; 150.1; 149.5; 149.1; 147.0; 143.8; 142.1; 141.8; 141.4;141.0; 139.6; 131.8; 128.0 (2 carbons); 127.7; 127.6; 126.9; 126.8 (2carbons); 123.1; 118.7; 116.4; 73.9; 67.4; 58.7; 58.3; 55.5; 48.2; 41.2;37.7; 34.1; 25.9; 18.5; 18.0; 12.0; H) a specific optical rotation [α]²⁰_(D) of +182.5 in CHCl₃ +10% CH₃ OHwhich comprises subjecting antibioticGE 2270 factor A to a hydrolysis reaction: 1) with an aqueous solutionor suspension of a mineral acid in which said mineral acid is selectedfrom the group consisting of hydrogen chloride, hydrogen bromide,hydrogen iodide, sulfuric acid and phosphoric acid, or with an aqueoussolution or suspension an organic acid in which said organic acid isselected from the group consisting of C₁ -C₆) aliphatic acids,halogenated(C₂ -C₅) aliphatic acids, arylic acids, (C₂ -C₆)alkylsulfonic acids, and arylic sulfonic acids; 2) in the presence of a polarorganic solvent selected from the group consisting of lower alkanols,phenyl substituted lower alkanols, lower alkyl carboxamides, lower alkylsulfoxamides, lower alkyl phosphoramides, lower alkyl sulfoxides, loweralkyl sulfones and cyclic oxygen containing aliphatic solvents; 3) at atemperature ranging from 40° C. to 90° C.; 4) for a period of timeranging from 8 hours to 48 hours, and; 5) monitoring the reaction forthe appearance antibiotic GE 2270 factor A₃ and recovering saidantibiotic GE 2270 factor A₃ from the reaction medium.